Immunization is the cornerstone of seasonal influenza control and represents an important component of pandemic preparedness strategies. Using a bioluminescent reporter H1N1pdm09 virus, we demonstrate the application of non-invasive in vivo imaging system (IVIS) technology to evaluate the pre-clinical efficacy of candidate vaccines and immunotherapy in a mouse model of influenza. Sequential imaging revealed distinct spatiotemporal kinetics of bioluminescence between groups of mice that were passively or actively immunized by various strategies that accelerated clearance of challenge virus at different rates and by distinct mechanisms. Imaging findings were consistent with conclusions derived from virus titration from the lungs and, notably, were more informative than conventional efficacy endpoints in some cases. Our findings demonstrate the reliability of IVIS as a qualitative approach to support pre-clinical evaluation of candidate medical countermeasures for influenza in mice.